The present invention relates generally to tissue regeneration, e.g., the treatment of wounds using a growth factor- or angiogenic factor-secreting cells admixed with a biological or synthetic extracellular matrix and/or attached/applied to a wound dressing or solid nondegradable support matrix.
Wounds (i.e., lacerations or openings) in mammalian tissue can result in tissue disruption and coagulation of the microvasculature at the wound face. Repair of such tissue represents an orderly, controlled cellular response to injury. All soft tissue wounds, regardless of size, heal in a similar manner. The mechanisms of tissue growth and repair are biologic systems wherein cellular proliferation and angiogenesis occur in the presence of an oxygen gradient. The sequential morphological and structural changes, which occur during tissue repair have been characterized in great detail and have, in some instances, been quantified. See Hunt, T. K., et al., xe2x80x9cCoagulation and macrophage stimulation of angiogenesis and wound healing,xe2x80x9d in The surgical wound, pp. 1-18, ed. F. Dineen and G. Hildrick-Smith (Lea and Febiger, Philadelphia: 1981).
Tissue regeneration in various organs, such as, e.g., the skin or the heart depends on connective tissue restoring blood supply and enabling residual organ-specific cells such as keratinocytes or muscle cells to reestablish organ integrity. Thus, a relevant function of the mesenchymal cells, i.e., the fibroblasts or, in addition, the endothelial cells of vasculature, is secretion of factors enhancing the healing process, e.g., factors promoting formation of new blood vessels (angioneogenesis) or factors promoting re-epithelialization by proliferating and migrating keratinocytes.
The cellular morphology of a wound consists of three distinct zones. The central avascular wound space is oxygen deficient, acidotic and hypercarbic, and has high lactate levels. Adjacent to the wound space is a gradient zone of local anemia (ischemia), which is populated by dividing fibroblasts. Behind the leading zone is an area of active collagen synthesis characterized by mature fibroblasts and numerous newly formed capillaries (i.e., neovascularization). While new blood vessel growth (angiogenesis) is necessary for the healing of wound tissue, angiogenic agents generally are unable to fulfill the long-felt need of providing the additional biosynthetic effects of tissue repair. Despite the need for more rapid healing of wounds (i.e., severe burns, surgical incisions, lacerations and other trauma), to date there has been only limited success in accelerating wound healing with pharmacological agents.
The primary goal in the treatment of wounds is to achieve wound closure. Open cutaneous wounds represent one major category of wounds. This category includes acute surgical and traumatic, e.g., burn wounds, as well as chronic wounds such as neuropathic ulcers, pressure sores, arterial and venous (stasis) or mixed arterio-venous ulcers, and diabetic ulcers. Open cutaneous wounds routinely heal by a process comprising six major components: i) inflammation, ii) fibroblast proliferation, iii) blood vessel proliferation, iv) connective tissue synthesis, v) epithelialization, and vi) wound contraction. Wound healing is impaired when these components, either individually or as a whole, do not function properly. Numerous factors can affect wound healing, including malnutrition, infection, pharmacological agents (e.g., cytotoxic drugs and corticosteroids), diabetes, and advanced age. See Hunt et al., in Current Surgical Diagnosis and Treatment (Way; Appleton and Lange), pp. 86-98 (1988).
Skin wounds, which do not readily heal can cause the subject considerable physical, emotional, and social distress as well as great financial expense. See e.g., Richey et al., Annals of Plastic Surgery 23(2):159-65 (1989). Indeed, wounds that fail to heal properly finally may require more or less aggressive surgical treatment, e.g., autologous skin grafting. A number of treatment modalities have been developed as scientists"" basic understanding of wounds and wound healing mechanisms has progressed.
The most commonly used conventional modality to assist in cutaneous wound healing involves the use of wound dressings. In the 1960s, a major breakthrough in wound care occurred when it was discovered that wound healing with a moist occlusive dressings was, generally speaking, more effective than the use of dry, non-occlusive dressings. See Winter, Nature 193:293-94 (1962). Today, numerous types of dressings are routinely used, including films (e.g., polyurethane films), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (cross-linked polymers containing about at least 60% water), foams (hydrophilic or hydrophobic), calcium alginates (nonwoven composites of fibers from calcium alginate), and cellophane (cellulose with a plasticizer). See Kannon et al., Dermatol. Surg. 21:583-590 (1995); Davies, Burns 10:94 (1983). Unfortunately, certain types of wounds (e.g., diabetic ulcers, pressure sores) and the wounds of certain subjects (e.g., recipients of exogenous corticosteroids) do not heal in a timely manner (or at all) with the use of such dressings.
Several pharmaceutical modalities have also been utilized in an attempt to improve wound healing. For example, treatment regimens involving zinc sulfate have been utilized by some practitioners. However, the efficacy of these regimens has been primarily attributed to their reversal of the effects of sub-normal serum zinc levels (e.g., decreased host resistance and altered intracellular bactericidal activity). See Riley, Am. Fam. Physician 24:107 (1981). While other vitamin and mineral deficiencies have also been associated with decreased wound healing (e.g, deficiencies of vitamins A, C and D; and calcium, magnesium, copper, and iron), there is no strong evidence that increasing the serum levels of these substances above their normal levels actually enhances wound healing. Thus, except in very limited circumstances, the promotion of wound healing with these agents has met with little success.
What is needed is a safe, effective, and interactive means for enhancing the healing of extensive and/or hard-to-heal wounds that can be used without regard to the type of wound or the nature of the patient population.
The present invention relates to the use of angiogenic or other growth factors expressed by human cells in unencapsulated pastes (mixed with matrix material or synthetic biocompatible substances) to be temporarily applied to wounds or defects in skin or other tissues for the restoration of blood supplying connective tissue to enable organ-specific cells to reestablish organ integrity as well as to inhibit excessive scar formation.
In one aspect, the invention involves a cell paste for tissue regeneration, e.g., in the treatment of skin wounds containing a cell or a combination of cell types that secrete biologically active substances, admixed with an extracellular matrix material such that the admixture forms a viscous cell paste.
In various embodiments, the cells are stromal, epithelial or organ specific, or a blood-derived cell, such as a fibroblast, a keratinocyte (including outer root sheath cells), a melanocyte, an endothelial cell, a pericyte, a monocyte, a lymphocyte (including plasma cells), a thrombocyte, a mast cell, an adipocyte, a muscle cell, a hepatocyte, a neuron, a nerve or neuroglia cell, an osteocyte, an osteoblast, corneal epithelial cells, chondrocyte, and/or an adult or embryonic stem cell. Preferably, the cells of this invention are allogeneic or xenogenic. Preferably, the cells are differentiated allogenic fibroblasts and keratinocytes.
The main cell type of connective tissue is the fibroblast. Until recently, fibroblasts have been dealt with like homogenous non-differentiating cell populations. However, the fibroblast cell system in various species, including man, is a stem cell system in which the fibroblasts terminally differentiate along seven stages, three containing mitotic and four including post-mitotic cells. See Bayreuther et al., Proc. Natl. Acad. Sci. USA 85:5112-16 (1988); Bayreuther et al., J. Cell. Sci. Suppl. 10:115-30 (1988). In vitro induction of fibroblast differentiation may be performed by chemical or biological agents, such as mitomycin C (Brenneisen et al., Exp. Cell. Res. 211:219-30 (1994)) or growth factors or cytokines (Hakenjos et al., Int. J. Radiat. Biol. 76:503-09 (2000)) such as TGF beta 1, IL-1, IL-6, Interferon alpha. In vitro induction may also be accomplished by irradiation with, e.g., X-rays (Bumann et al., Strahlenther. Onkol. 171:35-41 (1995); UV light (Rodemann et al., Exp. Cell. Res. 180:84-93 (1989); or physical exposure to electromagnetic fields (Thumm et al., Radiat. Environ. Biophys. 38:195-99 (1999). Moreover, induction of differentiation may also be accomplished by culture conditions such as serum starvation or contact inhibition. See Palka et al., Folia Histochem. Cytobiol. 34:121-27 (1996).
To date, the function/biological properties of differentiated fibroblasts have been poorly studied. The pattern of polypeptide expression and secretion, however, varies from mitotic to post-mitotic stages. The respective polypeptides are still being analyzed. See, e.g., Francz, Eur. J. Cell. Biol. 60:337-45 (1993).
In other embodiments, the biologically active molecule is an angiogenic factor or a growth factor, or a combination of at least one angiogenic factor and at least one growth factor. Examples of suitable biologically active molecules include, but are not limited to, epidermal growth factor-growth factor family (EGF); transforming growth factor alpha; HGF/SF; Heparin-binding epidermal growth factor; basic fibroblast growth factor; acidic fibroblast growth factor; other fibroblast growth factors; keratinocyte growth factor; transforming growth factors xcex21 and xcex22; transforming growth factor xcex23; platelet derived growth factor; vascular endothelial growth factor; tumor necrosis factor; interleukin-1 and -6; other interleukin/cytokine family members; insulin-like growth factor I; colony-stimulating factor 1; GM-CSF; and PDGF. Those skilled in the art will recognize that additional biologically active molecules can also be used in the methods and compositions of the invention.
In various embodiments, the extracellular matrix or matrix material can be collagens, alginate, alginate beads, agarose, fibrin, fibrin glue, blood plasma fibrin beads, whole plasma or components thereof, laminins, fibronectins, proteoglycans, HSP, chitosan, heparin, and/or other synthetic polymer scaffolds and solid support materials that could hold or adhere to cells such as wound dressings.
In a further embodiment, the cells are mitotically inactivated, i.e., induced to various stages of differentiation. For example, this inactivation can be accomplished by the administration of mitomycin C or other chemically-based mitotic inhibitors, irradiation with xcex3-Rays, irradiation with X-Rays, or irradiation with UV light.
In a still further embodiment, the cells are genetically engineered to secrete an exogenous level of angiogenic factors or growth factors. This secretion may be constitutive. Alternatively, this secretion may be controlled by gene switching.
In various other embodiments, the invention also provides methods of treating tissue defects or wounds by administering the cell pastes according to the invention to a wound site on a patient in need of wound treatment.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.